PROJECT SUMMARY Pancreatic ?-cells secrete insulin to maintain glucose homeostasis. The secretory activity of ?-cells is under the control of a constellation of ion channels. The long-term goal of this project is to understand how ion channels are regulated in ?-cells and how these regulations impact insulin secretion in health and disease. For the past 15 years of this project we have focused exclusively on the ATP-sensitive potassium (KATP) channel, which plays a key role in coupling glucose metabolism to ?-cell excitability. In this application, we have expanded our focus to include the voltage-gated delayed rectifier Kv2.1 channels, which are critical for repolarizing ?-cell membrane potential following action potential firing. This new focus is based on our recent finding that KATP and Kv2.1 channel densities in the ?-cell membrane are dynamically and concurrently regulated by the adipocyte-derived hormone leptin, which is known to suppress insulin secretion. We showed that leptin selectively recruits KATP and Kv2.1 channels to the ?-cell membrane via a common signaling mechanism dependent on the AMP-activated protein kinase (AMPK), cAMP-dependent protein kinase (PKA), and actin depolymerization. The goal of this renewal application is to build on the new, exciting research direction we have developed and further elucidate the signaling and cellular mechanisms by which leptin regulates trafficking of ?-cell KATP and Kv2.1 channels. Based on compelling preliminary data we hypothesize that leptin regulates trafficking of KATP and Kv2.1 channels in a concerted manner via a signaling mechanism involving the NMDA-subtype ionotropic glutamate receptor (NMDA receptor), the Ca2+/calmodulin dependent kinase CaMKK?, AMPK, PKA, and actin remodeling to achieve coordinated control of insulin secretion, and that this regulatory mechanism is lost in type 2 diabetic ?-cells. Three interrelated specific aims are proposed to test the hypothesis. (1) Elucidating the role of NMDA receptors in KATP and Kv2.1 channel trafficking regulation in ?-cells. (2) Mapping the leptin signaling network that regulates KATP and Kv2.1 channel trafficking, in particular focusing on dissecting the relationship between AMPK, PKA, and their downstream targets. (3) Identifying the subcellular vesicular localizations of KATP and Kv2.1 channels that underlie trafficking co-regulation by leptin. We will employ an integrated research design combining state-of-the-art electrophysiology, imaging, and proteomics techniques using both a ?-cell line model as well as primary human ?- cells from healthy and diabetic donors, to accomplish these aims. The research is innovative in testing the novel concept that trafficking regulation of KATP and Kv2.1 channels by leptin is a physiological mechanism to control insulin secretion and that defects in this mechanism may contribute to the pathogenesis of type 2 diabetes. As such, the research is highly significant to both basic science and human health. Successful outcome will significantly advance the scientific knowledge on this relatively new topic in the ?-cell biology/diabetes research field. The knowledge obtained will enable us to manipulate the system to better control insulin secretion and overcome metabolic diseases related to unbalanced insulin/leptin secretion or signaling.